Bottom Line:
Alternatively, Cx43alpha1 may serve a novel function in motility.We observed that p120 catenin (p120ctn), an Armadillo protein known to modulate cell motility, is colocalized not only with N-cadherin but also with Cx43alpha1.Moreover, the subcellular distribution of p120ctn was altered with N-cadherin or Cx43alpha1 deficiency.

ABSTRACTConnexin 43 (Cx43alpha1) gap junction has been shown to have an essential role in mediating functional coupling of neural crest cells and in modulating neural crest cell migration. Here, we showed that N-cadherin and wnt1 are required for efficient dye coupling but not for the expression of Cx43alpha1 gap junctions in neural crest cells. Cell motility was found to be altered in the N-cadherin-deficient neural crest cells, but the alterations were different from that elicited by Cx43alpha1 deficiency. In contrast, wnt1-deficient neural crest cells showed no discernible change in cell motility. These observations suggest that dye coupling may not be a good measure of gap junction communication relevant to motility. Alternatively, Cx43alpha1 may serve a novel function in motility. We observed that p120 catenin (p120ctn), an Armadillo protein known to modulate cell motility, is colocalized not only with N-cadherin but also with Cx43alpha1. Moreover, the subcellular distribution of p120ctn was altered with N-cadherin or Cx43alpha1 deficiency. Based on these findings, we propose a model in which Cx43alpha1 and N-cadherin may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cell's locomotory apparatus through p120ctn signaling.

fig6: Immunolocalization of p120ctn in N-cadherin– and Cx43α1-deficient neural crest cells. Neural crest cells from wild-type, Cx43α1, and N-cadherin knockout mouse embryos were fixed in the presence of Triton X-100 and then immunostained with a p120ctn antibody. In wild-type neural crest cells, p120ctn localized predominantly at the cell periphery, outlining regions of cell–cell contact found in cell processes (A and B). In such Triton extracted cells, some nuclear localization of p120ctn can be observed (A). In neural crest cells from the Cx43α1-deficient embryos (C), p120ctn expression in the cell periphery is reduced, and there is increased localization of p120ctn in the nucleus. In the N-cadherin–deficient neural crest cells (D), p120ctn expression at the cell periphery is generally not observed except in some cell processes. However, there is very strong localization of p120ctn in the nucleus. These images were obtained by standard immunofluorescence microscopy. All images are at the same magnification. Bar, 25 μm.

Mentions:
To further investigate the mechanism by which N-cadherin and Cx43α1 gap junctions may modulate neural crest cell motility, we considered the potential role of p120ctn, an Armadillo protein which binds to the juxtamembrane region of cadherin and is kown to modulate cell motility through the Rho GTPases (for review see Anastasiadis and Reynolds, 2000). Studies using various tissue culture cells have shown that p120ctn can be found distributed between the cell membrane, cell cytoplasm, and the nucleus (Anastasiadis and Reynolds, 2000). Analysis by immunohistochemistry showed that p120ctn is expressed abundantly in mouse cardiac neural crest cells. It is membrane localized at regions of cell–cell contact, and it is also present diffusely in the cell cytoplasm (Fig. 5 B). When the neural crest cells were fixed in the presence of 0.3% Triton X-100 to extract the cytoplasmic p120ctn, localizaton of p120ctn in the nucleus also can be discerned (Fig. 5 E and Fig. 6 A). The level of nuclear staining varied somewhat between explants with most being at very low levels (Fig. 5 E and Fig. 6 B). However, in 1 of 15 wild-type neural crest explants examined a more prominent level of nuclear staining was observed (Fig. 6 A). The inclusion of Triton in the fixative also made the membrane-bound p120ctn more sharply delineated, even when viewed by standard immunofluorescence microscopy (see Fig. 5 E, with Triton treatment; compare with B, without Triton treatment). This is presumably due to the removal of cytoplasmic p120ctn. It is important to note that the overall pattern of p120ctn distribution at the cell surface in Triton-treated cells was identical to that seen in cells fixed without Triton, being associated mostly with regions of cell–cell contact. Note that Fig. 5 B shows a relatively low level of cytoplasmic p120ctn immunostaining, since the image was obtained from optical slices of only 0.2 μm in thickness. In contrast, Fig. 5 E is a standard immunofluorescent image, but most of the cytoplasmic p120ctn has been extracted due to the inclusion of Triton during fixation.

fig6: Immunolocalization of p120ctn in N-cadherin– and Cx43α1-deficient neural crest cells. Neural crest cells from wild-type, Cx43α1, and N-cadherin knockout mouse embryos were fixed in the presence of Triton X-100 and then immunostained with a p120ctn antibody. In wild-type neural crest cells, p120ctn localized predominantly at the cell periphery, outlining regions of cell–cell contact found in cell processes (A and B). In such Triton extracted cells, some nuclear localization of p120ctn can be observed (A). In neural crest cells from the Cx43α1-deficient embryos (C), p120ctn expression in the cell periphery is reduced, and there is increased localization of p120ctn in the nucleus. In the N-cadherin–deficient neural crest cells (D), p120ctn expression at the cell periphery is generally not observed except in some cell processes. However, there is very strong localization of p120ctn in the nucleus. These images were obtained by standard immunofluorescence microscopy. All images are at the same magnification. Bar, 25 μm.

Mentions:
To further investigate the mechanism by which N-cadherin and Cx43α1 gap junctions may modulate neural crest cell motility, we considered the potential role of p120ctn, an Armadillo protein which binds to the juxtamembrane region of cadherin and is kown to modulate cell motility through the Rho GTPases (for review see Anastasiadis and Reynolds, 2000). Studies using various tissue culture cells have shown that p120ctn can be found distributed between the cell membrane, cell cytoplasm, and the nucleus (Anastasiadis and Reynolds, 2000). Analysis by immunohistochemistry showed that p120ctn is expressed abundantly in mouse cardiac neural crest cells. It is membrane localized at regions of cell–cell contact, and it is also present diffusely in the cell cytoplasm (Fig. 5 B). When the neural crest cells were fixed in the presence of 0.3% Triton X-100 to extract the cytoplasmic p120ctn, localizaton of p120ctn in the nucleus also can be discerned (Fig. 5 E and Fig. 6 A). The level of nuclear staining varied somewhat between explants with most being at very low levels (Fig. 5 E and Fig. 6 B). However, in 1 of 15 wild-type neural crest explants examined a more prominent level of nuclear staining was observed (Fig. 6 A). The inclusion of Triton in the fixative also made the membrane-bound p120ctn more sharply delineated, even when viewed by standard immunofluorescence microscopy (see Fig. 5 E, with Triton treatment; compare with B, without Triton treatment). This is presumably due to the removal of cytoplasmic p120ctn. It is important to note that the overall pattern of p120ctn distribution at the cell surface in Triton-treated cells was identical to that seen in cells fixed without Triton, being associated mostly with regions of cell–cell contact. Note that Fig. 5 B shows a relatively low level of cytoplasmic p120ctn immunostaining, since the image was obtained from optical slices of only 0.2 μm in thickness. In contrast, Fig. 5 E is a standard immunofluorescent image, but most of the cytoplasmic p120ctn has been extracted due to the inclusion of Triton during fixation.

Bottom Line:
Alternatively, Cx43alpha1 may serve a novel function in motility.We observed that p120 catenin (p120ctn), an Armadillo protein known to modulate cell motility, is colocalized not only with N-cadherin but also with Cx43alpha1.Moreover, the subcellular distribution of p120ctn was altered with N-cadherin or Cx43alpha1 deficiency.

ABSTRACTConnexin 43 (Cx43alpha1) gap junction has been shown to have an essential role in mediating functional coupling of neural crest cells and in modulating neural crest cell migration. Here, we showed that N-cadherin and wnt1 are required for efficient dye coupling but not for the expression of Cx43alpha1 gap junctions in neural crest cells. Cell motility was found to be altered in the N-cadherin-deficient neural crest cells, but the alterations were different from that elicited by Cx43alpha1 deficiency. In contrast, wnt1-deficient neural crest cells showed no discernible change in cell motility. These observations suggest that dye coupling may not be a good measure of gap junction communication relevant to motility. Alternatively, Cx43alpha1 may serve a novel function in motility. We observed that p120 catenin (p120ctn), an Armadillo protein known to modulate cell motility, is colocalized not only with N-cadherin but also with Cx43alpha1. Moreover, the subcellular distribution of p120ctn was altered with N-cadherin or Cx43alpha1 deficiency. Based on these findings, we propose a model in which Cx43alpha1 and N-cadherin may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cell's locomotory apparatus through p120ctn signaling.